Docosahexaenoic acid disrupts in vitro amyloid β1‐40 fibrillation and concomitantly inhibits amyloid levels in cerebral cortex of Alzheimer’s disease model rats

We have previously reported that dietary docosahexaenoic acid (DHA) improves and/or protects against impairment of cognition ability in amyloid beta_1‐40 (Aβ_1‐40)‐infused Alzheimer’s disease (AD)‐model rats. Here, after the administration of DHA to AD model rats for 12 weeks, the levels of Aβ_1‐40, cholesterol and the composition of fatty acids were investigated in the Triton X100‐insoluble membrane fractions of their cerebral cortex. The effects of DHA on the in vitro formation and kinetics of fibrillation of Aβ_1‐40 were also investigated by thioflavin T fluorescence spectroscopy, transmission electron microscopy and fluorescence microscopy. Dietary DHA significantly decreased the levels of Aβ_1‐40, cholesterol and saturated fatty acids in the detergent insoluble membrane fractions of AD rats. The formation of Aβ fibrils was also attenuated by their incubation with DHA, as demonstrated by the decreased intensity of thioflavin T‐derived fluorescence and by electron micrography. DHA treatment also decreased the intensity of thioflavin fluorescence in preformed‐fibril Aβ peptides, demonstrating the anti‐amyloidogenic effects of DHA. We then investigated the effects of DHA on the levels of oligomeric amyloid that is generated during its in vitro transformation from monomers to fibrils, by an anti‐oligomer‐specific antibody and non‐reducing Tris‐Glycine gradient (4–20%) gel electrophoresis. DHA concentration‐dependently reduced the levels of oligomeric amyloid species, suggesting that dietary DHA‐induced suppression of in vivo Aβ_1‐40 aggregation occurs through the inhibitory effect of DHA on oligomeric amyloid species.     

Conformation‐specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen

We engineered and employed a chaperone‐like amyloid‐binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross‐reacted with amyloid beta‐peptide (Aβ42) protofibrils, but not with Aβ40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1‐hIAPP complex cross‐react with Aβ42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation‐specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation‐sensitive and sequence‐independent and can target more than one type of protofibril species.     

Diabetes exacerbates amyloid and neurovascular pathology in aging‐accelerated mice

Mounting evidence supports a link between diabetes, cognitive dysfunction, and aging. However, the physiological mechanisms by which diabetes impacts brain function and cognition are not fully understood. To determine how diabetes contributes to cognitive dysfunction and age‐associated pathology, we used streptozotocin to induce type 1 diabetes (T1D) in senescence‐accelerated prone 8 (SAMP8) and senescence‐resistant 1 (SAMR1) mice. Contextual fear conditioning demonstrated that T1D resulted in the development of cognitive deficits in SAMR1 mice similar to those seen in age‐matched, nondiabetic SAMP8 mice. No further cognitive deficits were observed when the SAMP8 mice were made diabetic. T1D dramatically increased Aβ and glial fibrillary acidic protein immunoreactivity in the hippocampus of SAMP8 mice and to a lesser extent in age‐matched SAMR1 mice. Further analysis revealed aggregated Aβ within astrocyte processes surrounding vessels. Western blot analyses from T1D SAMP8 mice showed elevated amyloid precursor protein processing and protein glycation along with increased inflammation. T1D elevated tau phosphorylation in the SAMR1 mice but did not further increase it in the SAMP8 mice where it was already significantly higher. These data suggest that aberrant glucose metabolism potentiates the aging phenotype in old mice and contributes to early stage central nervous system pathology in younger animals.     

Disrupted‐in‐schizophrenia‐1 protects synaptic plasticity in a transgenic mouse model of Alzheimer’s disease as a mitophagy receptor

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD). Accumulated damaged mitochondria, which are associated with impaired mitophagy, contribute to neurodegeneration in AD. We show levels of Disrupted‐in‐schizophrenia‐1 (DISC1), which is genetically associated with psychiatric disorders and AD, decrease in the brains of AD patients and transgenic model mice and in Aβ‐treated cultured cells. Disrupted‐in‐schizophrenia‐1 contains a canonical LC3‐interacting region (LIR) motif (²¹⁰FSFI²¹³), through which DISC1 directly binds to LC3‐I/II. Overexpression of DISC1 enhances mitophagy through its binding to LC3, whereas knocking‐down of DISC1 blocks Aβ‐induced mitophagy. We further observe overexpression of DISC1, but not its mutant (muFSFI) which abolishes the interaction of DISC1 with LC3, rescues Aβ‐induced mitochondrial dysfunction, loss of spines, suppressed long‐term potentiation (LTP). Overexpression of DISC1 via adeno‐associated virus (serotype 8, AAV8) in the hippocampus of 8‐month‐old APP/PS1 transgenic mice for 4 months rescues cognitive deficits, synaptic loss, and Aβ plaque accumulation, in a way dependent on the interaction of DISC1 with LC3. These results indicate that DISC1 is a novel mitophagy receptor, which protects synaptic plasticity from Aβ accumulation‐induced toxicity through promoting mitophagy.     

Amyloid precursor protein 695 associates with assembled NR2A‐ and NR2B‐containing NMDA receptors to result in the enhancement of their cell surface delivery

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co‐immunoprecipitated with assembled NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1‐1a, NR1‐2a, NR1‐4b^c‐Myc, or NR2 subunit transfections revealed that co‐association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2′ cassettes and, the use of an NR1‐2a^c‐Myc‐trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N‐terminal domains. Anti‐APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti‐NR1 or anti‐NR2A antibodies co‐immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co‐expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer’s disease.     

Nurr1 (NR4A2) regulates Alzheimer’s disease‐related pathogenesis and cognitive function in the 5XFAD mouse model

The orphan nuclear receptor Nurr1 (also known as NR4A2) is critical for the development and maintenance of midbrain dopaminergic neurons, and is associated with Parkinson's disease. However, an association between Nurr1 and Alzheimer's disease (AD)‐related pathology has not previously been reported. Here, we provide evidence that Nurr1 is expressed in a neuron‐specific manner in AD‐related brain regions; specifically, it is selectively expressed in glutamatergic neurons in the subiculum and the cortex of both normal and AD brains. Based on Nurr1’s expression patterns, we investigated potential functional roles of Nurr1 in AD pathology. Nurr1 expression was examined in the hippocampus and cortex of AD mouse model and postmortem human AD subjects. In addition, we performed both gain‐of‐function and loss‐of‐function studies of Nurr1 and its pharmacological activation in 5XFAD mice. We found that knockdown of Nurr1 significantly aggravated AD pathology while its overexpression alleviated it, including effects on Aβ accumulation, neuroinflammation, and neurodegeneration. Importantly, 5XFAD mice treated with amodiaquine, a highly selective synthetic Nurr1 agonist, showed robust reduction in typical AD features including deposition of Aβ plaques, neuronal loss, microgliosis, and impairment of adult hippocampal neurogenesis, leading to significant improvement of cognitive impairment. These in vivo and in vitro findings suggest that Nurr1 critically regulates AD‐related pathophysiology and identify Nurr1 as a novel AD therapeutic target.     

SCFFbx2‐E3‐ligase‐mediated degradation of BACE1 attenuates Alzheimer’s disease amyloidosis and improves synaptic function

BACE1 (β‐secretase) plays a central role in the β‐amyloidogenesis of Alzheimer’s disease (AD). The ubiquitin–proteasome system, a major intracellular protein quality control system, has been implicated recently in BACE1 metabolism. We report that the SCF^Fbx2‐E3 ligase is involved in the binding and ubiquitination of BACE1 via its Trp 280 residue of F‐box‐associated domain. Physiologically, we found that Fbx2 was expressed in various intracellular organelles in brain neurons and that BACE1 is colocalized with Fbx2 and the amyloid precursor protein (APP), mainly at the early endosome and endoplasmic reticulum. The former are believed to be the major intracellular compartments where the APP is cleaved by BACE1 and β‐amyloid is produced. Importantly, we found that overexpression of Fbx2 in the primary cortical and hippocampal neurons derived from Tg2576 transgenic mice significantly promoted BACE1 degradation and reduced β‐amyloid production. In the search for specific endogenous modulators of Fbx2 expression, we found that PPARγ coactivator‐1α (PGC‐1α) was capable of promoting the degradation of BACE1 through a mechanism involving Fbx2 gene expression. Interestingly, we found that the expression of both Fbx2 and PGC‐1α was significantly decreased in the brains of aging Tg2576 mice. Our in vivo studies using a mouse model of AD revealed that exogenous adenoviral Fbx2 expression in the brain significantly decreased BACE1 protein levels and activity, coincidentally reducing β‐amyloid levels and rescuing synaptic deficits. Our study is the first to suggest that promoting Fbx2 in the brain may represent a novel strategy for the treatment of AD.